An important field of diagnostics is the use of rapid immunodiagnostic assays to provide speed, accuracy and simplicity in the diagnosis and testing in subjects, such as testing for diseases, conditions, microbes or drugs. A common form of such an assay is a lateral flow immunoassay, which is commonly employed in devices such as pregnancy test kits.
Lateral flow immunoassays are widely used for self-testing and in the clinical setting in view of their simplicity, speed and reliability, and involve a non-electrical method for rapidly detecting the presence of a specific analyte in a liquid sample, for example as described in United States Patent Application No. 2005/0227371.
Lateral flow immunoassays generally involve applying a liquid sample suspected of containing a predetermined analyte onto a porous carrier, and the liquid sample then traverses the porous carrier by capillary action. Different porous materials can be used for the porous carrier, and may differ in aspects such as pore size, wicking or flow rate, protein-binding aspects and pre-treatment. Essentially, all of the physical activities and chemical reactions take place in the porous carrier. The liquid sample is applied onto a sampling-end of the porous carrier (e.g. ‘proximal end’ or ‘wet end’) for a measured time or volume (e.g. 5 seconds or 2 drops). The liquid sample then migrates along the porous carrier by capillary action to the ‘distal’ or ‘dry’ end. The liquid sample can be pre-treated for optimized reaction with additional agents e.g. pH agents or buffers, surfactants, and/or blockers, which are typically impregnated into the porous carrier. The analytes in the sample can be ‘labelled’ for detection by using a labelled reagent (e.g. ‘detection reagent’) that has affinity for binding to a predetermined analyte. The sample can be labelled before contact with the porous carrier, or alternatively the porous carrier can include a ‘labelling zone’ where the sample mobilizes a labelled reagent that has been reversibly (temporarily) immobilized in the porous carrier. While the analyte is reacting with the mobilized labelled reagent, the liquid sample and mobilized labelled reagent migrates further within the porous carrier to a detection zone (e.g. ‘capture zone’) where a capture reagent (e.g. immobilised capture antibody) that binds the same analyte is immobilized to the porous carrier, usually in the form of a line. When analyte is present in the liquid sample, a ‘sandwich’ in the form of the labelled reagent:analyte:capture antibody is formed, and the resulting concentration of the labelled reagent leads to a detectable line appearing in the detection zone, which indicates a positive result. Any remaining sample liquid, together with the rest of the labelled reagent continues to migrate to a control zone and/or porous sink. Unbound labelled reagent that has not reacted with the predetermined analyte, and which remains in the porous carrier, contributes to a background signal that can reduce detection accuracy.
Nitrocellulose membranes are typically used in lateral flow immunoassays as a porous carrier material. However, some variability exists in nitrocellulose membrane materials arising from processes for preparing the materials, which can result in reduced accuracy and precision of tests. This variability in producing nitrocellulose membranes, which results in variation in wicking rates, causes a reproducibility problem where lateral flow tests have traditionally performed poorly for quantitative measurement, with assay coefficients of variability (CV) being commonly in the range 20-40%, such as described in J Agric. Food Chem. 2012 Nov. 21; 60(46):11491-7 and Anal. Chim. Acta. 2013 Apr. 15; 772:75-80. An assay CV of 25% means that the 95% confidence interval for a test result is the mean+/−50%. Such poor imprecision is not suitable for accurate measurements, particularly for quantitative measurement in determining the concentration of a target analyte in a sample, and on which clinical decisions may be based. An incorrect diagnosis may lead to incorrect clinical decision-making which may in turn lead to adverse health outcomes. Although other types of porous materials have been used as alternatives to nitrocellulose membrane materials, they also typically suffer from poor imprecision, particularly where analyte detection methods are reliant on low background noise.
A range of methods can be used for labelling an analyte and detecting the presence of a labelled analyte in a sample, for example colorimetric labels, radioisotopes and fluorescent labels, which have binding affinity for the predetermined analyte, may be used. For example, labelling using colorimetric latex beads has been described in U.S. Pat. No. 5,451,504. Conventional lateral flow tests using visual markers (such as colloidal gold labels) are known to perform poorly in terms of sensitivity. Other labelling techniques can also be problematic when used in rapid diagnostic assays for detecting small quantities of particular analytes in samples. Fluorescent labels have been used within some types of immunoassay systems, but their sensitivity has been typically limited by background fluorescence of the naturally-fluorescing porous carriers and constituents thereof, or from the presence of unbound fluorescent labels.
Consequently, there is a need to identify alternative and improved lateral flow immunoassay devices and systems that are accurate, cost-effective and rapidly enable the detection of a target analyte in a sample.